The Receptor Interacting Protein Kinases (RIPK1 and RIPK3) are proteins involved in cell death and survival. The role of RIPK1 in regulating these outcomes is determined by context and the kinase versus scaffolding function of the protein. Necroptosis is a form of regulated cell death dependent on the kinase function of RIPK1, activating the RIPK1-RIPK3 necrosome leading to the activation of MLKL and cell membrane rupture when caspases are inhibited. Using the acetaminophen (APAP) liver injury model, we found no role for necroptosis as RIPK3-/- and MLKL -/- mice are not protected. However, we identified a novel scaffolding function for RIPK1 in APAP toxicity. We have shown that knocking down RIPK1 protects mice from APAP hepatotoxicity upstream of JNK, a key mediator of APAP induced cell death. Interestingly, our preliminary studies reveal RIPK1 kinase-dead transgenic mice are not protected from APAP, strongly suggesting a scaffolding function of the protein in cell death signaling. In addition, we have found a cell specific distribution for hepatic RIPK3 protein, with abundant protein expression in the non-parenchymal cells, including liver sinusoidal endothelial cells (LSECs). These findings have lead to the following overarching hypothesis, which forms the basis of this career development proposal in two clinically relevant liver injury models: APAP hepatotoxicity, and ischemia reperfusion injury (IRI). RIPK1 regulates cell death in the liver through a scaffolding function in hepatocytes and a kinase function in LSECs. For the first two years of the award we will focus on aim1, which will systematically explore the scaffolding function of RIPK1 in hepatocytes. We will also determine the interacting proteins with RIPK1 and the role of RIPK1 in the signaling pathway that leads to JNK activation in APAP toxicity and other ER stress models. Subsequently, in the second aim of the proposal we will explore the effect of cell type specific deletion of RIPK1 and RIPK3 in models of liver injury by examining liver injury in mice with conditional hepatocyte or endothelial deletions. The proposed studies are the core components of the Mentored Clinical Scientist Development Award (K08) for Dr. Lily Dara, around which a structured program for Dr. Dara has been built. Dr. Dara has identified four target areas for additional training throughout the K Award period: protein biochemistry and signaling, transgenic mice models, LSEC pathobiology, and the liver ischemia reperfusion injury model. To achieve these goals, she has devised a 5-year career development plan and assembled a multidisciplinary advisory team of scientists specializing in each of these core components to guide her work and critique her findings. The University of Southern California has a long-standing tradition in excellence in liver research. The NIDDK- funded USC Research Center for Liver Disease is an excellent resource for the candidate, providing both practical facilities and equipment through its cores, and the opportunity for scientific exchange and collaboration through its large membership base. The immediate focus of this project in the next five years is the role of RIPK1 and RIPK3 in models of acute liver injury. After completion of these studies the long-term goal of Dr. Dara is to extend these findings to experimental models of chronic liver injury such as alcoholic and nonalcoholic fatty liver disease/steatohepatitis. The experiments planned in this proposal, the guidance of the mentorship advisory committee, and the ideal training environment at USC will ensure Dr. Dara's successful transition to an independent physician-scientist while advancing the understanding of the role of RIP kinases in liver disease.